1. Field of the Invention
The present invention relates to a recombinant vector and a transgenic mouse for expressing human ferritin in a tissue non-specific manner, and more particularly, to a vector prepared by operably linking a human ferritin gene to a promoter including a cytomegalovirus (CMV) early enhancer element and a β-actin promoter, and a transgenic mouse for expressing human ferritin in a tissue non-specific manner, which is transformed, with, the vector. Further the present invention relates to a method for generating a transgenic mouse, and a method for monitoring cell or tissue therapy using the transgenic mouse.
2. Description of the Related Art
Ferritin is a bioactive molecule present in the liver, spleen, and bone marrow of mammals. Ferritin, first isolated from the spleen and liver of horse by Lauflberger in 1937, is composed of 24 protein subunits surrounding a central iron core (diameter: 120-130 A.U., inner cavity diameter: 75 A.U.) according to X-Ray and electron microscopy. That is, each ferritin molecule is composed of a spherical protein shell with an average molecular weight of 450,000, called apoferritin, which consists of 24 protein subunits with a molecular weight of approximately 19,000 and contains 20˜30% Fe as a ferric hydroxyphosphate polymer form. Thus, ferritin is a physiological active molecule as a sole source of iron which is released from the protein and utilized in the body when demanded. When fully loaded with Fe, ferritin can store up to 4,500 iron atoms per molecule, which is equivalent to an iron concentration capable of producing 1200 hemoglobin molecules.
Meanwhile, ferritin, the iron storage protein essential for life, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Ferritin is a metalloprotein containing iron atoms, and thus functions as a nanomagnet to be utilized as an MRI reporter. Relying on signals derived from hydrogen protons produced by water-molecules exposed to a magnetic field, regular MRI converts the signals into images. In ferritin-applied MRI, however, the nanomagnet directly stimulates surrounding protons to generate signals. Therefore, ferritin allows non-invasive MRI without injection of coutrast agents.
The ferritin heavy polypeptide subunit (FTH) has a potent, ferroxidase activity that catalyzes the oxidation of ferrous iron. The magnetic resonance (MR) properties of FTH were the focus of extensive research and showed abnormality with high relaxivity at very low iron loading on the magnetic field. The overexpression of FTH could augment R2 relaxivity upon magnetic resonance imaging (MRI) by redistribution of iron among more ferritin complexes as well as by increased total cellular iron level.
It was reported that the TET-mFTH transgenic mice overexpressing HA-tagged mouse FTH and EGFP (enhanced green fluorescent protein) were generated in a tissue specific and tetracycline inducible manner by using the MR properties of ferritin (Cohen, B., Ziv, K., Plaks, V., Israely, T., Kalchenko, V., Harmelin, A., Benjamin, L. E. and Neeman, M. (2007). MRI detection of transcriptional regulation of gene expression in transgenic mice, Nat Med 13, 498-503.). The above report suggested the possibility for MR application of ferritin as a reporter gene in multiple organs and the safety of chronic overexpression of FTH for applications for long-term tracking. According to the known technologies, however, ferritin cannot be applied to MR imaging of multiple organs because of its tissue specific expression, and there is also no report on the technologies of expressing the ferritin gene in a tissue non-specific manner.
The present inventors have made an effort to find a technology for expressing the ferritin gene in a tissue non-specific manner. As a result, they developed the transgenic mouse (C5BL/6-β-actin-hFTH) expressing human ferritin (hFTH) under the control of ubiquitous CAG promoter, which allows broader expression. The hFTH expression was detected in most tissue such as the brain, heart, liver, lung, spleen, pancreas and kidney by RT-PCR and Western blot. The histological changes in tissues with overexpression of hFTH were not detected. They evaluated hFTH expression as a functional MR reporter by performing noninvasive magnetic resonance imaging (MRI) of transgenic mice using 9.4 T. Expression of the hFTH in the brain and liver tissues of mice leads to a significant decrease in T2* relaxation time. Based on this study, the present inventors also demonstrated that hFTH can be used as an MR reporter and the transgenic mice of the present invention would be an available model of hFTH-based molecular imaging to study potential therapies for the cell and tissue graft, thereby completing the present invention.